Topical antimicrobial compositions and methods of using same

ABSTRACT

The invention includes a method of treating a mammal comprising topically applying an aqueous composition to a target site on the mammal, wherein the aqueous composition comprises: active ingredients comprising i) a cosmetic dye selected from a violet, blue or green dye, or combinations thereof, and ii) an enhancing ingredient, wherein the relative weight percentage of the cosmetic dye to the enhancing ingredient is about 1:2 to about 40:1, wherein the cosmetic dye interacts with keratinous material thereby i) substantially staining the target site and ii) inhibiting the active ingredients from significantly leaching from the target site.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.61/336,074, filed Jan. 14, 2010, and U.S. application Ser. No.12/930,786, filed Jan. 14, 2011, both of which are incorporated hereinby reference in their entireties.

BACKGROUND OF THE INVENTION

The present invention relates to compositions and methods of providingrapid and persistent control of a broad-spectrum of microbes onmammalian surfaces by topical application of compositions.

Lameness of diary cows and sheep is one of the major problems facing thedairy and meat industry, respectively, today. The cost of lameness ismeasured by lost milk production, culled cows, underweight meat animals,additional labor, veterinary bills, and medicines for treatment. In theUnited States, the annual overall cost of lameness is estimated toexceed 570 million dollars. It is especially a problem in large herds,which are the fastest growing segment of the market. The prevalence oflameness in large herds is 50% or more.

Lameness is, in many cases, caused by anaerobic organisms such asArcanobacterium pyogenes (previously called Corynebacterium pyogenes),in particular where the infection goes into the deep structure of thehoof. In sheep the infection may be accompanied by swelling and a whiteto black pus discharge.

There are multiple causes for lameness ranging from lack of generalstable hygiene, specific hoof hygiene, hoof care, malnutrition, generalgenetic disposition, specific genetic defects to bacterial and viralinfections. For example, fibroma (corns) is a genetic condition found incattle which causes a hard, fibrous lump to form between the claws ofthe foot. The corn makes the hoof more susceptible to subsequentbacterial and viral infections. These infections occur at variouslocations in and around the hooves of the cattle.

In addition to the general lesions caused by infection, a disease ofunknown etiology has been spreading throughout the western UnitedStates. This disease is digital dermatitis. Digital dermatitis ischaracterized by painful lesions, which often are surrounded by a ridgeof hyperkeratotic (thickened) skin with finger like projections. Due toits appearance, the disease is commonly referred to as hairy wart orstrawberry disease; it is also known as Mortellaro disease.

Studies have shown that a third of all lameness in cows is caused bydigital dermatitis. Digital dermatitis is present worldwide and isestimated to be present in 41% of herds smaller than 100 cows, and from64% to 82% in larger herds.

Other contagious and debilitating diseases of the bovine foot and hoofinclude such conditions as bovine hoof rot and interdigital dermatitis.In addition to causing lameness in diary cows, these contagious diseasesalso cause a significant decrease in milk production and sometimesdeath. Dairy farmers report as much as 50% less milk being produced percow. (Linda Leake, Farm Journal, Inc., (1998).) In sheep, in addition tocausing lameness and reduced production, foot rot is highly contagioussometimes resulting in a whole flock becoming infected. (Government ofNewfoundland and Labarador 2004)

For years, directing animals through hoof baths containing hoof caresolutions has been practiced as an aid to general hoof health; andhygiene for the prevention of, and a cure for, diseases of the animals'hooves. Materials used for these solutions include copper sulfate, zincsulfate, formaldehyde, quaternary ammonium compounds, peroxides, organicacids, and their combinations, and certain antibiotics. Additionally,several over the counter commercially compounded liquid products havebeen and are being used. Major manufacturers of this latter category areDelaval (DoubleAction), Westfalia-Surge (Pedicure Rx), and SSICorporation (Healthy Foot). Copper sulfate, zinc sulfate and other heavymetal based antimicrobials are usually applied in 5% to 10% solutions tobe effective.

Recently, the negative environmental impact of the use of heavy metalcompounds has been recognized. Efforts have been made to use mixed saltsin combination with specific additives in order to reduce the overalluse of heavy metal (to concentrations as low as low as 0.5%). However,despite regulatory restrictions, heavy metals are still in use since noeffective alternative products are presently on the market.

Hoof baths are generally located in the return alley of dairy milkingbarns or parlors. After being milked, the animals typically walk throughthe hoof bath on the way back to where they are housed. The feet andhooves will typically contain accumulated dirt and manure. This isespecially true in modern dairy facilities with housing contained inlimited areas such as free stall or tie stall barns or dry lots insteadof open pasture.

In addition, on passing through the hoof bath, the cows may defecateinto the hoof bath. The added organic material or load to the hoof bathcompromises the antimicrobial products' ability to work in thedisinfection and cleansing of the cow hooves where the causativemicroorganisms are located. For economic reasons, the use ofantibacterial chemical and biological products in doses high enough tocompensate for the organic material present in the hoof bath and topenetrate through organic material and whatever tissue may conceal orotherwise harbor the bacterial pathogens, is usually cost prohibitive.Other chemical products that are less expensive to use at higher doseshave the disadvantage in that they may be toxic to the animals, to thepeople working in the dairy facilities, and, particularly, to theenvironment. Heavy metal moieties of the compounds do not break down andaccumulate in the environment.

Accordingly, there is a need for a more effective and environmentallyfriendly manner by which to treat and prevent hoof diseases in bovinelivestock. In particular, a need exists for an effective method tocontrol a broad spectrum of microbes that is fast-acting, haslong-lasting efficacy, and is mild to livestock.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a method of treating amammal comprising topically applying an aqueous composition to a targetsite on the mammal, wherein the aqueous composition comprises: activeingredients comprising i) a cosmetic dye selected from a violet, blue orgreen dye, or combinations thereof, and ii) an enhancing ingredient,wherein the cosmetic dye interacts with keratinous material thereby i)substantially staining the target site and ii) inhibiting the activeingredients from significantly leaching from the target site.

The relative weight percentage of the cosmetic dye to the enhancingingredient is about 1:2 to about 40:1. In one embodiment, the relativeweight percentage of the cosmetic dye to the enhancing ingredient isabout 10:1. The relative weight percentage of the active ingredients towater is about 1:5 to about 1:1000.

In one embodiment, the mammal is bovine livestock. The treatment of thelivestock includes inhibiting disease, preventing disease, assisting inhealing lesions, maintaining or improving hygiene, or combinationsthereof. In one instance, the target site is the hoof the livestock. Thelivestock can be treated for hoof rot, foot rot, digital dermatitisand/or interdigital dermatitis. Typically, the livestock is treated bycontacting the hoof of the livestock with a hoof bath comprising theaqueous composition. In another embodiment, the target site is the teatof the livestock and the disease is mastitis.

In one embodiment, the cosmetic dye is Gentian Violet, Brilliant Green,Toluidine Blue, or combinations thereof.

In one embodiment, the enhancing ingredient is selected from the groupconsisting of halogenated isothiazolin-3-ones; formaldehyde depotsubstances; chloracetamide; hexetidine; O-phenylphenol;2,4-dichlorobenzylalcohol; trichlorcarban; glyoxal; chlorocresol, sodiumhydroxymethylglycinate; sodium 2-biphenylate, chlorhexidine digluconate;chlorhexidine diacetate; hexamidine; phenoxyethanol; biphenyl-2-ol,formic acic, benzoic acid, salicylic acid, lactic acid, tannic acid,symclosene, sodium dichloroisocyanurate dehydrate, sorbic acid, methylparaben; bronopol; triclosan; chlorhexidine; chlorhexidine digluconate,5-isopropyl-2-methylphenol; 4-chloroxylol; DMDM-hydantoine; chlorophene,chloramin-T, benzylalcohol; cyanamide, phenoxyisopropanol;dimethyloxazolidine; benzylhemiformal; silver chloride, chlorobutanol;imazalil, sodium p-chloro-m-cresolate, diamine, troclosene sodium,phenol; herbal extracts, thymol, menthol, rosemary oil, carvacrol,magnolia bark extract and synthetics.

In one aspect, the present invention provides a topical aqueouscomposition comprising: active ingredients comprising i) a cosmetic dyeselected from a violet, blue or green dye, or combinations thereof, andii) at least one enhancing ingredient, wherein the relative weightpercentage of the cosmetic dye to the enhancing ingredient is about 2:1to about 40:1, wherein the cosmetic dye interacts with keratinousmaterial.

In one aspect, the present invention provides a topical aqueouscomposition comprising: i) water, and ii) active ingredients wherein theactive ingredients consist essentially of an cosmetic dye selected froma violet, blue or green dye, or combinations thereof, and at least oneenhancing ingredient, wherein the relative weight percentage of thecosmetic dye to the enhancing ingredient is about 1:2 to about 40:1,wherein the cosmetic dye interacts with keratinous material, and whereinthe relative weight percentage of the active ingredients to water isabout 1:5 to about 1:1000.

In one aspect, the present invention provides a dry compositioncomprising: active ingredients comprising i) a cosmetic dye selectedfrom a violet, blue or green dye, or combinations thereof, and ii) atleast one enhancing ingredient, wherein the relative weight percentageof the cosmetic dye to the enhancing ingredient is about 2:1 to about99:1, wherein the cosmetic dye interacts with keratinous material in thepresence of water.

In another aspect, the present invention provides a method of treating abovine mammal comprising: exposing the mammal to an aqueous composition.The aqueous composition comprises active ingredients comprising i) acosmetic dye selected from a violet, blue or green dye, or combinationsthereof, and ii) at least one enhancing ingredient; and water. Therelative weight percentage of the cosmetic dye to the enhancingingredient is about 2:1 to about 99:1. The relative weight percentage ofthe active ingredients to water is about 1:5 to about 1:1000. Thecosmetic dye interacts with keratinous material of the mammal. The dyeprovides the composition with a color, wherein the lost of the colorsignifies the lost of effectiveness of the aqueous composition. As anexample, the aqueous composition is a hoof bath.

In a further aspect, the present invention provides a method of treatinga bovine mammal comprising: placing a dissoluble pouch containing a drycomposition into a hoof bath. The dry composition comprises: activeingredients comprising i) a cosmetic dye selected from a violet, blue orgreen dye, or combinations thereof, and ii) at least one enhancingingredient, wherein the relative weight percentage of the cosmetic dyeto the enhancing ingredient is about 2:1 to about 99:1. The relativeweight percentage of the active ingredients to water is about 1:5 toabout 1:1000.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the influence of hoof bath contamination on the compositionof the present invention (at 2% and 4% concentrations) versus coppersulfate (at 2.5% and 5% concentrations) after 4 hrs, 24 hrs, 48 hrs and96 hrs.

FIG. 2 shows the influence of hoof bath contamination on the compositionof the present invention (at 2% and 4% concentrations) versusformaldehyde (at 2% concentration) after 4 hrs, 24 hrs and 48 hrs.

FIG. 3 shows the influence of hoof bath contamination on the compositionof the present invention (at 2% and 4% concentrations) versusglutaraldehyde (at 2% concentration) after 4 hrs, 24 hrs and 48 hrs.

FIG. 4 shows the influence of hoof bath contamination on the compositionof the present invention (at 2% and 4% concentrations) versusbenzalkonium chloride (at 1% concentration) after 4 hrs, 24 hrs, 48 hrsand 72 hrs.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides aqueous concentrate compositionsconveniently suitable for dilution into a use concentration, and the enduse compositions; and methods to topically treat mammalian microbialdisease and infection, and lesions and wounds resulting therefrom.

The present invention provides dry compositions conveniently suitablefor dilution into use concentrations, and methods to topically treatmammalian microbial disease and infection, and lesions and woundsresulting therefrom, with such compositions. The dry compositions aresubstantially (e.g., entirely) free of moisture, e.g., free of water.

The compositions and methods of the present invention enable killing abroad spectrum of bacteria, including Gram positive and Gram negativebacteria, while simultaneously inactivating or destroying viruses andfungi. The compositions are particularly effective against anaerobicbacteria, for example, Arcanobacterium pyogenes. The compositions arefast-acting and provide long-lasting efficacy.

Throughout this specification, quantities are defined by ranges, and bylower and upper boundaries of ranges. Each lower boundary can becombined with each upper boundary to define a range. The lower and upperboundaries should each be taken as a separate element.

In one embodiment, the active ingredients of the compositions of thepresent invention (including the aqueous, concentrate, end use and drycompositions) comprise i.) a violet, green or blue cosmetic dye, orcombinations thereof, and ii.) at least one cosmetic preservative and/orantimicrobial (hereinafter “enhancing ingredient”).

The cosmetic dye comprises a cationic nitrogen, wherein the cationicnitrogen is connected to a double bond of an aromatic carbon. Thecationic nitrogen has bonds to only three carbon atoms, including onedouble bond to an aromatic carbon. This double bond to the cationicnitrogen can oscillate from one to all the other possible cationicnitrogen atoms in the molecule giving the molecule the color.

Examples of suitable violet, green and blue cosmetic dye include GentianViolet, Brilliant Green and Toluidine Blue. These dyes can be4-[(4-dimethylaminophenyl)-phenyl-methyl]-N,N-dimethyl-aniline; and(1-amino-8-methyl-phenothiazin-3-ylidene)-dimethyl-ammonium. Furtherexamples include Brilliant Blue FCF, Ethyl Green, Green S, and VictoriaBlue BO.

It has surprisingly been discovered that at a certain relative amount ofthe cosmetic dye to the enhancing ingredient, the antimicrobial efficacyof the component parts of the compositions of the present invention aresynergistically enhanced.

The preferred relative weight percentage of the cosmetic dye to theenhancing ingredient has a range of about 1:2 to about 40:1 (or of about2:1 to about 40:1). Examples of other preferred lower boundaries of thisrange include 5:1; 10:1, 15:1 and 20:1. Examples of other preferredupper boundaries of this range include 20:1; 25:1; 30:1 and 35:1.

Other examples of preferred relative weight percentage of the cosmeticdye to the enhancing ingredient has a range of about 90:1 to about 99:1.Examples of other preferred boundaries of this range include 95:1; and97:1. Examples of other preferred ranges are about 95:5 to and 98.9:1.As specific examples, the relative weight percentage of the cosmetic dyeto the enhancing ingredient is about 98.894:0.1057 or about 98.8:0.2.For example, the enhancing ingredient can be present in about 0.2 toabout 5 wt. % of the composition. Also, in some embodiments, thecomposition of the present invention can consist essentially of (orconsist of) only the cosmetic dye.

In the aqueous compositions of the present invention, the relativeweight percentage of the active ingredients to water is about 1:5 toabout 1:1000, or about 1:25 to about 1:1000. Examples of other preferredlower boundaries of this range include about 1:10; about 1:100; 1:200,1:400 and 1:500. Examples of other preferred upper boundaries of thisrange include 1:500; 1:750; and 1:900.

For example, in a preferred embodiment, the aqueous composition containsapproximately a maximum of 3 wt. % active ingredients and 97 wt. %water. For instance, the active ingredients can be 0.31 wt. % of theaqueous composition, of which 0.2 wt. % is the violet, green or bluecosmetic dye and 0.01 wt. % is the enhancing ingredient and 0.1% is astabilizer. Accordingly, in such example, the corresponding amount ofwater is about 99.69 wt. % so that the actives to water ratio is about0.31:99.69 or 1:322.

In a preferred example, about one pound of a dry composition of thepresent invention is added to about 50 gallons of water to form an enduse composition.

In some embodiments, the compositions of the present invention canfurther comprise a gelling and/or thickening agent wherein the ratio ofthe active ingredients to the agent is about 2:1 to 1 to 40. Examples ofsuch agents include gums, xanthem gum, cellulose, methylcellulose,carboxymethylcellulose, chitosan, alginates, polysaccharides, theirderivatives and combinations thereof.

In a preferred embodiment, the compositions do not include quaternaryammonium compounds. (In quaternary ammonium compounds, the cationicnitrogen has four separate bonds to four carbon atoms.) Also, in apreferred embodiment, the compositions also do not include and/or do notinclude an anticoagulant and/or do not include peroxides and/or do notinclude heavy metal based antimicrobials. Some examples ofanticoagulants include heparin, hirudin, EGTA, urokinase, streptokinase,and hydrogen peroxide. Some examples of heavy metal based antimicrobialsinclude copper sulfate and zinc sulfate. In a preferred embodiment, thecompositions do not include cosmetic preservatives based on an organicacid except for tannic acid. The compositions also preferably do notinclude harsh chemicals, such as, for example, formaldehyde and/orglutaraldehyde, and/or do not contain antibiotics.

Accordingly, the synergistic combination of the i) cosmetic dye and ii)the enhancing ingredient(s) decrease the minimum inhibitoryconcentration (MIC) of each individual component. Also, the activeingredients of the present invention are effective at a much lowerconcentration than the active ingredients of other antimicrobialcompositions which use dyes or cosmetic preservative compounds; and areeffective at a much lower concentration than heavy metal basedantimicrobials.

Additionally, the combination of the cosmetic dye and the enhancingingredient(s) increase the spectrum of microbes that each componentcould individually target. The compositions of the present inventionenable killing a broad spectrum of bacteria, including Gram positive andGram negative bacteria such as S. aureus, S. choleraesuis, E. coli, K.pneumoniae, and anaerobic bacteria, such as, for example,Arcanobacterium pyogenes while simultaneously inactivating or destroyingviruses and fungi. The cosmetic dyes suitable for the compositions aredyes which interact (e.g., bind) with keratinous material when actingsynergistically with the enhancing ingredients of the present invention.

Further, once the compositions of the present invention contain water,the compositions degrade. Thus, the compositions are environmentallyfriendly and do not interfere with the operation of the digester.

The topical antimicrobial composition can be in the form of aconcentrate for ease of transport. Preferably, in the concentrate, andconsequently in the diluted ready-to-use form, the active ingredients towater are at about 1:5 to 1:500 or 1:1000. A preferred concentrate hasabout 10 wt. % dye and about 2 wt. % specific cosmetic preservative.Such a concentration makes shipping very economically. Further examplesinclude a concentrate having a ratio of about 1:9 (actives to water). Inthe diluted form, the composition has a typical ratio of about 0.2 to99.8, that is, of about 2 to 998 or about 1:500. Typical other ready touse ratios include about 1:100 or 1:200 or about 1:300, up to about1:1000.

The dry compositions of the present invention are also in a form whichallows ease of transport. Typically, dry composition comprises about 0.2to about 5 wt. % of a dry enhancing ingredient (e.g., crystalline form)with the balance being substantially the cosmetic dye in dry form.

The aqueous compositions (i.e. use compositions) of the presentinvention are fast-acting and long-lasting. In particular, the aqueouscompositions quickly penetrate porous keratinous material (e.g.,keratinous material of skin tissue, hooves, finger/toe nails, and hair).Once the site of interest (i.e., the target site) is in contact with thecomposition for few seconds, the cosmetic dye interacts with thekeratinous material of a mammal in such a way that it is resistant toleaching.

Because the composition is resistant to leaching, the duration of theantimicrobial activity is increased. For example, at least about 90%,about 80%, about 70%, about 60%, about 50%, or about 40% of thecomposition remains at the target site despite exposure to percolatingwater. This property of the composition is very beneficial when appliedto bovine livestock since such animals are typically exposed toenvironmental water, for example, while grazing.

Additionally, the cosmetic dye substantially stains the target site.Such staining is a useful property of the compositions. For example, thedye indicates the presence of the active ingredients, and theirpenetration depth; that is, the protected zone is clearly elucidated.Using this indicator feature of the composition, it has beendemonstrated that even after extensive exposure to percolating water,the depth of penetration of the composition and its intensity ofstaining are not diminished.

In one embodiment, the active ingredients of the present inventionconsists essentially of (or consists of) a cosmetic dye and at least oneenhancing ingredient. That is, other ingredients that may materiallyaffect the basic and novel characteristics of the active ingredients ofthe invention are specifically excluded from the composition. The activeingredients are the cosmetic dye(s) and the enhancing ingredient(s) ofthe compositions of the invention (e.g., aqueous composition), or theprimary antimicrobial ingredients of the compositions of the invention(e.g., aqueous composition). Preferably, such composition also includesa stabilizer. An example of this embodiment is the dry compositions ofthe present invention.

A typical pH range of the aqueous compositions of the present inventionis from about 2.5 to 7. A typical pH range for a concentrate of thecompositions is about 2.8 to 3. The end user typically may make about 2%to 5% aqueous solution for a hoof bath. Such a solution typically has apH of slightly below about 3.5. In actual use, when cows walk through a2% hoof bath and drop manure, the pH typically gradually rises to aboveabout 4. A pH of below about 7 does not typically decrease the efficacyof the compositions.

Methods of Use

In another embodiment, the present invention includes a method oftreating mammals by topically applying the compositions to desiredtarget sites. In this specification “treating” refers to inhibitingdisease; preventing disease; aiding in the prevention of disease;assisting in healing lesions; maintaining or increasing the hygienelevel, or combinations thereof. Typical target sites include sites onthe body that are susceptible to microbial infection and are prone tolesions and wounds. The diseases which can be treated by the methods ofthe present invention include any microbial infections; lesions orwounds that result from such infections; and lesions or wounds that arevulnerable to such infections.

In embodiments where compositions of the present invention are in theform of a gel, the gel is dabbed onto a target site, and optionally, abandage may be applied.

The invention is particularly well suited for treating bovine mammals,in particular, bovine livestock. Examples of bovine mammals includecows, cattle, and ox, The compositions are also suitable for ovine,caprine, hircine and corvine animals, such as, for example, sheep, goat,deer, yak, buffalo, antelope, bison, deer and elk. Examples of diseaseswhich can be treated include diseases related to hooves, including, forexample, hoof rot, digital dermatitis, interdigital dermatitis, footrot, strawberry disease (Mortellaro disease) and/or bacterial, viral andfungal infections.

Another disease of dairy animals that can be treated by the methods ofthe invention is mastitis. Mastitis is one of the most common andeconomically costly diseases confronting milk producers. Economic lossesresult from poor milk quality, lower milk production, and potentialculling of chronically infected animals.

The method of applying the composition depends on the disease and thetarget sites. For example, the composition can be sprayed, brushed,dabbed, or flooded onto the susceptible sites, such as, for example,hooves, skin, hair, and the udder.

One common application mode for hoof diseases is a hoof bath or a footbath. For example, the composition can be used in such a way thatanimals walk through the composition, thereby preventing the spread ofmicroorganisms, and also providing an opportunity to treat anyinfections on the hooves of the animals. Alternatively, the compositioncan be formulated and applied such that farm workers walk through thecomposition and thereby prevent microorganisms on their boots fromspreading.

In a preferred embodiment, a dry formulation of the composition isplaced into a dissolvable container, e.g., a pouch, a bag. Examples ofsuch containers include polyvinyl alcohol bags. Such bags are availablein a variety of grades, e.g., cold, warm and hot water soluble. Thecontainer holding the dry composition is placed into a hoof bath. Thecontainer and the dry composition dissolve quickly into the hoof bath.

For example, a pouch containing about one pound of a dry composition ofthe present invention can be added to 50 gallons of water in a hoofbath. One such pouch can be used for about 250 cows.

The cosmetic dye provides the bath with a color. For example, brilliantgreen provides the bath with a uniform blue color. Once the bath losesits color (e.g., its blue color), the bath is no longer effective. Also,the hooves are stained with the dye. Once the hooves lose the dye color,the hooves are no longer protected. Thus, the methods of the presentinvention provide an easily discernable visual cue signaling whether thecompositions are still effective.

The compositions of the present invention have a biodegradation time ofabout two weeks. Thus, hoof/foot baths using these compositions inconjunction with biogasgenerators is quite valuable, since they do notimpair the biogas generation. Copper sulfate is not suitable for suchuse because of its long term influence on the digester system stoppingthe biogas production, in addition to their negative environmentalimpact.

One common application mode for treating mastitis is a teat dip. Thecomposition is placed in a small container with a shape adapted to theteat. The teat is then dipped into the container filled with thecomposition. Another application mode is spraying the udder includingthe teats.

“Enhancing Ingredients”

Preferably, the enhancing ingredients include cosmetic preservative(s)and/or antimicrobials, such as, for example, halogenatedisothiazolin-3-ones; formaldehyde depot substances; chloracetamide;hexetidine; O-phenylphenol; 2,4-dichlorobenzylalcohol; trichlorcarban;glyoxal; sodium hydroxymethyl-glycinate; chlorhexidine digluconate;chlorhexidine diacetate; hexamidine; phenoxyethanol; methyl paraben;bronopol; triclosan; chlorhexidine; 5-isopropyl-2-methylphenol;4-chloroxylol; DMDM-hydantoine; benzylalcohol; phenoxyisopropanol;dimethyloxazolidine; benzylhemiformal; chlorobutanol; phenol; and herbalextracts and/or synthetics, for example, thymol, menthol, rosemary oil,carvacrol, and the like.

Other example of preferred enhancing ingredients (i.e., specificcosmetic preservative(s) and/or antimicrobials) is/are chlorocresol;sodium 2-biphenylate; biphenyl-2-ol; formic acid; benzoic acid;salicylic acid; lactic acid; tannic acid; symclosene; sodiumdichloroisocyanurate dehydrate; sorbic acid; chlorhexidine digluconate;chlorophene; chloramin-T; cyanamide; silver chloride; imazalil; sodiump-chloro-m-cresolate; diamine; troclosene sodium; and magnolia barkextract.

Further examples include polycarboxylic acid (e.g., malic acid, tartaricacid, citric acid, glutaric acid); saturated organic aliphatic monocarbonic acids; alpha/beta mono carbonic acids (e.g., hydroxyl inalpha/beta position, keto in the alpha/beta position); aromaticmonocarbonic acids, including those with substituents in the rings; andmandelic acid and its derivatives. These organic acids are provided incrystalline form in the dry compositions.

Auxiliary Substances

In addition to the active components, the above mentioned compositionscan include other, non-toxic auxiliary agents, as long as such agents donot detract from the benefits provided by the present therapeuticcompositions. These agents can, for example, facilitate the delivery andefficiency of the therapeutic agent and/or stabilize the composition(e.g., cosmetic stabilizers) with respect to its shelf life or itsactual outdoor applications. The preferred range of these agents in thecomposition is about 0.05% to 12%.

For example, these compositions can contain water-soluble skinconditioning or moisturizing agents that do not interfere with thesynergistic antimicrobial properties of the compositions. Examples ofthese ingredients are glycerin; glycols; polyols, such as polyethyleneglycol; lanolin; aloe vera, grapefruit seed extract, and vitamins, suchas E, C and A. These agents serve to assist in soothing and retainingmoisture on the skin. Examples of stabilizing agents (i.e., stabilizers)include cosmetic stabilizers; radical scavengers; antioxidants; and UVabsorbers e.g., cinnamate derivatives, benzophenone derivatives,vitamins and the like.

Agents such as colorants, fragrances and insect repellants (e.g.,citronella) may also be included in the composition. Other examples ofagents include preservatives, excipients, pH buffering agents, alcohols,chelating agents (e.g., EDTA), film-forming or barrier forminghydrophilic binder combinations (e.g. polyurethanes and polyvinylpyrrolidone) or other therapeutics that do not contaminate meat or dairyproducts produced by the animal, and mixtures thereof.

Carrier agents can also be included in the aqueous composition of thepresent invention. However, typically, the composition is used withoutbeing placed on, or in, a substrate. For example, the composition is notplaced onto a polyurethane polymer and/or not used in moldable/pliablecompositions. An example of a moldable/pliable composition is acomposition which comprises flour.

EXAMPLES

Digital dermatitis, mastitis and other related microbial infections inanimals can effectively be treated by a topical application of anaqueous formulation containing specific combinations of antimicrobialdyes and specific cosmetic preservative compound(s). The generalcomposition comprises, or consists essentially of, the following:

Example 1

-   0.001 to 2 wt. % of Gentian Violet-   0.001 to 0.1% of triclosan-   0.01 to 0.2 wt % of UV absorber;-   Rest Water.

Example 2

-   0.001% to 1% Brilliant Green;-   0.001% to 1% Toluidine blue-   0.005% to 0.5% Phenoxyethanol;-   Rest water.

Example 3

Sprayable sanitizer teat dip:

-   0.05 to 0.5 wt. % Brilliant Green;-   0.005% to 0.05 wt. % Phenoxyethanol;-   3% to 15 wt. % Ethanol;-   0.01% to 0.8 wt. % Polyurethane;-   0.005% to 0.3 wt. % Polyvinyl pyrrolidone;-   0.1% to 0.3 wt. % Yellow 5 Dye.-   Rest water

Example 4

-   0.05 to 0.5 wt. % Brilliant Green;-   0.005% to 0.05 wt. % Phenoxyethanol;-   Rest water

MIC Test against Enterococcus hirae according to JACh 48 (2001)

The minimum inhibiting concentration of the combination solution againstE. hirae was determined to be less than 0.05% with a 100% of organismreduction. Growth was observed at 0.005%. In conclusion the MIC isexpected to be between 0.005% to 0.05%

Example 5

MIC Test of example 4 against Arcanobacterium pyogenes (ATCC#9731 andATCC#19411) under anaerobic conditions were conducted by the TOXIKON. A100% reduction and a log-reduction of 6.63 was found at theconcentration of <0.0098% for both strains.

Example 6 Comparative Test

Under the same conditions as mentioned in Example 5 conducted by TOXIKONthe MIC of a 10% copper sulfate solution (commonly used in hoof bathsworldwide) was determined. Against Arcanobacterium pyogenes, StrainATCC#9731 and ATCC#19411 also under anaerobic conditions it was foundthat the 10% copper sulfate solution at the 8-times concentration, thatmeans it has kill rate of close to 100% when the concentration is atleast 0.078%.

Conclusion: In order to control the anaerobic organism Arcanobacteriumpyogenes, a required concentration of at least 8-times of a 10% coppersolution is needed in comparison to a solution according to example 4.

Example 7 Comparative Example

Hoof samples were submerged for 15 seconds in (1) the composition of theinvention (i.e., 4:1 of cosmetic dye:Phenoxyethanol, total amount about0.3 wt. %); (2.1) a 10 wt. % copper sulfate (2.2) a 10 wt. % zincsulfate solution; (3) untreated as a control. The samples were thenwashed for two hours to simulate leaching. The samples were then placedinto melted PA agar to solidify. Melted agar containing 10E5 CFU/ml ofT. mentagrophytes was then added and incubated at room temperature for 3and 6 days. The copper and zinc sulfate treated samples, as well as thecontrols, showed intense microbial growth. The hoof sample treated withthe composition of the invention had a clear zone of inhibition, about12 mm in radius and fewer microbial colonies around the hoof.

Example 7A Comparative Example

Hoof samples were submerged for 15 seconds in (1) the composition of theinvention (i.e., 4:1 of antimicrobial dye:Phenoxyethanol, total amountabout 0.21 wt. % or 0.3 wt. %); (2) a 10 wt. % copper sulfate or 10 wt.% zinc sulfate solution; or (3) untreated as a control. The samples werethen washed for two hours to simulate leaching. The samples were thenplaced into melted PA agar to solidify. Melted agar containing 10E5CFU/ml of T. mentagrophytes was then added and incubated at roomtemperature for 3 and 6 days. The Copper/Zinc sulfate treated samples,as well as the controls, showed intense microbial growth. The hoofsample treated with the composition of the invention had a clear zone ofinhibition, about 12 mm in radius and fewer microbial colonies aroundthe hoof.

Example 8

-   0.01% to 2.1% Brilliant Green;-   0.005% to 0.5% Benzylalcohol;-   99.985% to 97.4% water.

Example 9

-   0.01% to 2.1% Brilliant Green;-   0.005% to 0.5% Phenoxyethanol;-   0.01% to 0.2% Benzophenone 4;-   99.985% to 97.4% water.

Example 10

-   0.01% to 2.1% Brilliant Green;-   0.005% to 0.5% Bronopol;-   0.01% to 0.2% Ethylhexyl Methoxycinnamate;-   99.985% to 97.4% water.

Example 11

-   0.01% to 2.1% Brilliant Green;-   0.005% to 0.5% Glyoxal;-   0.01% to 0.2% Hydrochinone;-   99.985% to 97.4% water.

Example 12

-   0.05% Brilliant Green-   0.1% Phenoxyethanol-   0.2% Gentian Violet

Example 13 Comparative Antimicrobial Testing of Example 4 with CosmeticDyes According to Test Method MCR 3.0 with Actual Hoof Material

Solution of Example 4 showed good hoof penetration ability with 100%growth inhibition on the hoof against bacteria and 100% growthinhibition on the hoof against fungi.

Toluidine Blue in aqueous solution ranging from 0.25 to 0.5% also hadhigh hoof penetration ability but bacteria growth inhibition on the hoofwas only 80% and fungi growth inhibition on the hoof only 50%

Gentian Violet in aqueous solution ranging from 0.25 to 0.5% also showedhigh hoof penetration ability, but bacteria growth inhibition on thehoof was only 90% and fungi growth inhibition was only 80%

Methylene Blue also in aqueous solution of 0.25 to 0.5% showed high hoofpenetration ability but the growth inhibition against bacteria was only60% and against fungi only 50%.

Example 14

-   0.5% Toluidine Blue-   0.05% Brilliant Green-   Rest water

Formulation showed excellent hoof penetration but bacteria and fungigrowth inhibition was only 90%

Example 15

-   0.5% Methylene Blue-   0.05% Brilliant Green-   Rest water

Formulation showed excellent hoof penetration but only 60% bacteriagrowth inhibition and 80% fungi growth inhibition.

Example 16

-   0.5% Gentian Violet-   0.05% Brilliant Blue-   Rest Water

Formulation showed excellent hoof penetration and 100% growth inhibitionon the hoof against bacteria and fungi.

Example 17

-   0.5% Brilliant Green-   0.625% EDTA-   Rest water

Formulation showed good penetration into the hoof but only 80% growthinhibition on the hoof against bacteria and fungi.

Example 18

-   0.05% Brilliant Green-   Rest water

Formulation does not provide an inhibition activity on the hoof surface.

Example 19

-   0.05% Brilliant Green-   0.1% Phenoxyethanol-   0.2% Gentian Violet-   Rest water

The formulation was tested in comparison to formulation of Example 4 andcompared with an untreated control. The control showed no inhibitionagainst Escherichia coli, Staphylococcus aureus and fungi. Formulationof Example 4 showed complete inhibition against E. coli, S. aureus andfungi.

Formulation of Example 19 also showed complete inhibition against E.coli, S. aureus and fungi.

Example 20

-   0.5% Toluidine Blue-   Rest water

Formulation shows excellent penetration into the hoof in 15 sec butexhibits only 80% bacterial growth inhibition and 50% fungi growthinhibition.

Example 21

-   0.5% Toluidine Blue-   0.05% Grapefruit seed extract-   Rest water

Formulation shows excellent penetration into the hoof in 15 sec. Thebacteria and fungi growth inhibition was 100%

Example 22

-   0.1% Grapefruit seed extract-   Rest water

Formulation showed only 60% bacteria growth inhibition and 80% fungigrowth inhibition.

Example 23

According to standard method EN 14349 (Chemical disinfectants andantiseptics Quantitative surface test for the evaluation of bacterialactivity of chemicals disinfectants and antiseptics used in veterinaryfield on non-porous surfaces without mechanical action)

The Example 4 was tested against the Gram-negative organisms Pseudomonasaeruginosa and Proteus vulgaris as well as the Gram-positiveStaphylococcus aureus and showed in the eradicating application rate of5% a reduction rate of 10000 (4 log)

Example 24

-   1% Tannic acid-   0.2% Toluidine Blue-   Rest Water

Formulation showed no growth on a hoof surface where as 0.2% ToluidineBlue alone showed bacterial growth on a hoof surface.

Example 25

The objective was to evaluate the relative efficacy of a novel,commercially available disinfectant agent (T-Hexx Dragonhyde HBC,Hydromer, Branchburg, N.J.) compared with formalin and copper sulfate.The hypothesis was 2 sided; therefore, the hypothesis was that the newagent would be better or worse compared with the industry gold standardfootbath agents, formalin and copper sulfate. The study was conducted ina large commercial dairy farm. Two identical studies were conducted, thefirst comparing Dragonhyde (5% solution, twice weekly) and formalin (5%solution, twice weekly) and the second comparing Dragonhyde (5%solution, twice weekly) and copper sulfate (10% solution, twice weekly).The study design was identical for both studies with 4 pens (physicallyidentical), 2 treatments (Dragonhyde vs. formalin and Dragonhyde vs.copper sulfate), 2 periods (crossing over the treatment within pen), and3 repeated measures (3 observations per cow: enrollment, wk 2, and wk4). For study 1, 406 cows were enrolled (n=201 formalin and 205Dragonhyde). For study 2, 356 cows were enrolled (n=189 copper sulfateand 167 Dragonhyde). The adjusted odds of digital dermatitis lesion(DDL) throughout the study period were analyzed by mixed logisticregression model. In study 1, the odds of DDL were 1.36 times higher forthe formalin group compared with the Dragonhyde group. In study 2, thedata were analyzed by a similar statistical model and the variabletreatment did not significantly affect the overall prevalence of DDL. Inconclusion, the performance of 3 hoof care products was evaluated and itwas concluded that Dragonhyde performed better than formalin and thatthere was no difference between copper sulfate and Dragonhyde.

In one aspect, the present invention provides a method of treating amammal comprising topically applying an aqueous composition to a targetsite on the mammal, wherein the aqueous composition comprises activeingredients comprising an antimicrobial dye and specific cosmeticpreservative compound(s) (i.e., specific cosmetic preservative(s)). Therelative weight percentage of the antimicrobial dye to the specificcosmetic preservative(s) is about 2:1 to about 40:1. The antimicrobialdye interacts with keratinous material thereby i) substantially stainingthe target site and ii) inhibiting the active ingredients fromsignificantly leaching from the target site.

In one embodiment, the relative weight percentage of the antimicrobialdye to the specific cosmetic preservative(s) is about 20:1. In oneembodiment, the relative weight percentage of the active ingredients towater is about 1:25 to about 1:1000.

In one embodiment, the mammal is bovine livestock. The treatment of thebovine livestock comprises inhibiting disease, preventing disease,assisting in healing lesions, or combinations thereof. In one instance,the target site is the hoof the livestock. The livestock can be treatedfor hoof rot, digital dermatitis and/or interdigital dermatitis.Typically, the livestock is treated by contacting its hoof with a hoofbath comprising the aqueous composition. In another embodiment, thetarget site is the teat of the livestock and the disease is mastitis.

In certain embodiments, the antimicrobial dye can be a triarylmethanedye, a monoazo dye, a diazo dye, an indigoid dye, a xanthene or afluorescein dye, an anthraquinone dye, or a quinoline dye. Preferably,the antimicrobial dye is a triphenylmethane dye. Examples of preferredtriphenylmethane dyes includeN-[4[Bis[4-(dimethylamino)-phenyl]methylene]-2,5-cyclohexadien-1-ylidene]-N-methyl-methanaminiumchloride (i.e., Gentian Violet or Crystal Violet);N-[4-[[4-(Diethylamino)phenyl]-phenylmethylene]-2,5-cyclohexadien-1-ylidene]-N-ethylethanaminiumsulfate (1:1) (i.e., Brilliant Green); and Malachite Green.

The activity of triphenylmethane dyes is particularly enhanced by thesynergistic combination with the specific cosmetic preservatives of thepresent invention. In particular, triphenylmethane dyes are known tohave poor light stability and tend to be decolorized by bacteria (see,Jones, J. J. and Falkinham, J. III, Antimicrobial Agent andChemotherapy, 47(7):2323 (2003)). Also, a high concentration of thesetriphenylmethane dyes is typically needed to achieve the expectedfunctions due to their high minimum inhibition concentration (MIC).Moreover, triphenylmethane dyes are typically are only effective forGram positive bacteria. However, the synergistic combination with thespecific cosmetic preservatives enables triphenylmethane dyes toovercome their limitations. The triphenylmethane dyes become more lightstable, have a lower MIC, and become capable of inhibiting theactivities of Gram negative bacteria, viruses and fungi, in addition toinhibiting the activity of Gram positive bacteria.

In one embodiment the specific cosmetic preservative(s) is/arehalogenated isothiazolin-3-ones; formaldehyde depot substances;chloracetamide; hexetidine; O-phenylphenol; 2,4-dichlorobenzylalcohol;trichlorcarban; glyoxal; sodium hydroxymethylglycinate; chlorhexidinedigluconate; chlorhexidine diacetate; hexamidine; phenoxyethanol; methylparaben; bronopol; triclosan; chlorhexidine; 5-isopropyl-2-methylphenol;4-chloroxylol; DMDM-hydantoine; benzylalcohol; phenoxyisopropanol;dimethyloxazolidine; benzylhemiformal; chlorobutanol; phenol; and herbalextracts, for example, thymol, menthol, rosemary oil, carvacrol and thelike.

In one aspect, the present invention provides a topical aqueouscomposition comprising active ingredients comprising an antimicrobialdye and at least one specific cosmetic preservative wherein the relativeweight percentage of the antimicrobial dye to the specific cosmeticpreservative(s) is about 2:1 to about 40:1. The antimicrobial dyeinteracts with keratinous material. Preferably, the relative weightpercentage of the antimicrobial dye to the specific cosmeticpreservative(s) is about 20:1. Preferably, the relative weightpercentage of the active ingredients to water is about 1:25 to about1:1000. Preferably, the antimicrobial dye is a triphenylmethane dye.Preferably, the triphenylmethane dye is Gentian Violet, Brilliant Greenand Malachite Green.

Preferably, specific cosmetic preservatives include halogenatedisothiazolin-3-ones; formaldehyde depot substances; chloracetamide;hexetidine; O-phenylphenol; 2,4-dichlorobenzylalcohol; trichlorcarban;glyoxal; sodium hydroxymethylglycinate; chlorhexidine digluconate;chlorhexidine diacetate; hexamidine; phenoxyethanol; methyl paraben;bronopol; triclosan; chlorhexidine; 5-isopropyl-2-methylphenol;4-chloroxylol; DMDM-hydantoine; benzylalcohol; phenoxyisopropanol;dimethyloxazolidine; benzylhemiformal; chlorobutanol; phenol; and herbalextracts, for example, thymol, menthol, rosemary oil, carvacrol and thelike.

Additionally, the composition may contain cosmetic stabilizers, radicalscavengers, and UV absorbers e.g. cinnamate derivatives, benzophenonederivatives, vitamins and the like.

In one aspect, the present invention provides a topical aqueouscomposition comprising water and active ingredients wherein the activeingredients consist essentially of: an antimicrobial dye and at leastone specific cosmetic preservative. The relative weight percentage ofthe antimicrobial dye, to the specific cosmetic preservative(s) is about5:1 to about 40:1. The antimicrobial dye interacts with keratinousmaterial. The relative weight percentage of the active ingredients towater is about 1:25 to about 1:1000. Preferably, the relative weightpercentage of the antimicrobial dye to the specific cosmeticpreservative(s) is about 20:1.

Example 26

Dry Composition contains the following ingredients:

Dry Component Wt. % Raw Material Code Brilliant Green 98.894 RS0301Benzoic Acid 0.1057 RB-2000

The composition is placed into a one pound dissolvable pouch, and placedinto a 50 gallon hoof bath.

Example 27 Comparative Example

This example shows the influence of hoof bath contamination by dirt andmanure on the effectiveness of a composition of the present invention(at 2% and 4% concentrations) as compared with copper sulfate (at 2.5%and 5% concentrations). As can be seen in FIG. 1, the presentcomposition remains effective up to at least 48 hours aftercontamination; whereas, the control lose effectiveness even after 4hours.

Example 28 Comparative Example

This example shows the influence of hoof bath contamination by dirt andmanure on the effectiveness of a composition of the present invention(at 2% and 4% concentrations) as compared with formaldehyde (at 2%concentration). As can be seen in FIG. 2, the present compositionremains effective up to at least 48 hours after contamination; whereas,the control lose effectiveness even after 24 hours.

Example 29 Comparative Example

This example shows the influence of hoof bath contamination by dirt andmanure on the effectiveness of a composition of the present invention(at 2% and 4% concentrations) as compared with glutaraldehyde (at 2%concentration). As can be seen in FIG. 3, the present compositionremains effective up to at least 48 hours after contamination; whereas,the control lose effectiveness even after 24 hours.

Example 30 Comparative Example

This example shows the influence of hoof bath contamination by dirt andmanure on the effectiveness of a composition of the present invention(at 2% and 4% concentrations) as compared with benzalkonium chloride (at1% concentration). As can be seen in FIG. 4, the present compositionremains effective up to at least 72 hours after contamination; whereas,the control lose effectiveness even after 24 hours.

The invention claimed is:
 1. A method of treating a bovine mammalcomprising: contacting the hoof of the mammal in need thereof to a hoofbath consisting of: active ingredients consisting of i) a cosmetic dyeselected from Gentian Violet, Brilliant Green, Toluidine Blue, orcombinations thereof, and ii) benzoic acid; wherein the relative weightpercentage of the cosmetic dye to benzoic acid is about 90:1 to about99:1; and water, wherein the relative weight percentage of the activeingredients to water is about 1:100 to about 1:1000; and wherein thecosmetic dye interacts with keratinous material of the mammal, andwherein the dye provides the composition with a color, wherein the lossof the color signifies the loss of effectiveness of the hoof bath,wherein treating the bovine mammal consists of treating hoof rot, footrot, digital dermatitis, interdigital dermatitis; or combinationsthereof.
 2. The method of claim 1 wherein the mammal is bovinelivestock.
 3. The method of claim 1, wherein the dye is Brilliant Green.4. The method of claim 3, wherein the relative weight percentage ofBrilliant Green to benzoic acid is about 95:1 to about 99:1.
 5. A methodof treating a bovine mammal comprising: placing a dry composition intowater to form a hoof bath, contacting a hoof of the mammal in needthereof to the hoof bath, wherein the dry composition consists of:active ingredients consisting of i) a cosmetic dye selected from GentianViolet, Brilliant Green, Toluidine Blue, or combinations thereof, andii) benzoic acid; wherein the relative weight percentage of the cosmeticdye to benzoic acid is about 90:1 to about 99:1, wherein the relativeweight percentage of the active ingredients to water is about 1:100 toabout 1:1000; and wherein the cosmetic dye interacts with keratinousmaterial of the mammal, and wherein the dye provides the compositionwith a color, wherein the loss of the color signifies the loss ofeffectiveness of the hoof bath, wherein the dry composition is containedin a dissoluble pouch, wherein treating the bovine mammal consists oftreating hoof rot, foot rot, digital dermatitis, interdigitaldermatitis; or combinations thereof.
 6. The method of claim 5, whereinthe dye is Brilliant Green.
 7. The method of claim 6, wherein therelative weight percentage of Brilliant Green to benzoic acid is about95:1 to about 99:1.